Non-coding RNAs in Natural Killer/T-Cell Lymphoma

Natural killer/T-cell lymphoma (NKTCL) is a rare and aggressive subtype of non-Hodgkin's lymphoma that is associated with a poor outcome. Non-coding RNAs (ncRNAs), which account for 98% of human RNAs, lack the function of encoding proteins but instead have the important function of regulating gene expression, including transcription, translation, RNA splicing, editing, and turnover. However, the roles and mechanisms of aberrantly expressed ncRNAs in NKTCL are not fully clear. Aberrant expressions of microRNA (miRNAs) affect the PI3K/AKT signaling pathways (miRNA-21, miRNA-155, miRNA-150, miRNA-142, miRNA-494), NF-κB (miRNA-146a, miRNA-155) and cell cycle signaling pathways to regulate cell function. Moreover, Epstein-Barr virus (EBV) encoded miRNAs and EBV oncoprotein LMP-1 regulated the expression of cellular genes that induce invasion, metastasis, cell cycle progression and cellular transformation. In addition, NKTCL-associated Long non-coding RNA (lncRNA) ZFAS1 regulated certain pathways and lncRNA MALAT1 acted as a predictive marker. This review article provides an overview of ncRNAs associated with NKTCL, summarizes the function of significantly differentially expressed hotspot non-coding RNAs that contribute to the pathogenesis, diagnoses, treatment and prognosis of NKTCL and discusses the relevance of these ncRNAs to clinical practice.


INTRODUCTION
Non-Hodgkin's lymphoma (NHL) originates from B-lymphocytes, T-lymphocytes and natural killer (NK) lymphocytes and ranges from the indolent to the very aggressive (1). Each subtype could be further classified according to its origin, genetic signature or clinical features (2). Natural killer/T-cell lymphoma (NKTCL) is a rare and aggressive subtype of NHL that has a high incidence in East Asia and Latin America and that is associated with a poor outcome (3,4). Extranodal NKTCL can be further classified into nasal NKTCL, which primarily affects the nasal cavity, nasopharynx and the upper aerodigestive tract, and non-nasal NKTCL, which involves the outside of the nasopharyngeal region, such as the skin, gastrointestinal tract and testis (5).
Non-coding RNAs (ncRNAs), which account for 98% of all human RNAs, lack a protein-coding function, but rather, they have the important function of regulating gene expression, including transcription, translation, RNA splicing, editing, and turnover (13). NcRNAs include microRNAs (miRNAs), small nuclear RNAs, PIWI-interacting RNAs, long non-coding RNAs (lncRNA), and circular RNAs. With the development of nextgeneration sequencing and bioinformatics approaches, ncRNAs show great biological importance in cancers. However, the roles and mechanisms of aberrantly expressed non-coding RNAs in NKTCL have not been fully clarified. This review article provides an overview of the recent advancements of ncRNAs associated with NKTCL and discusses their relevance to clinical practice.

MiRNA-494
As a tumor suppressor miRNA, miRNA-494 played a role in various tumors (40,41). MiRNA-494 induced PTEN downregulation in cervical cancer cells (42) and myeloid cells (43). In addition, TGF-β1 was a tumor-derived factor that was associated with the upregulation of miRNA-494 in MDSCs and MMPs, which led to tumor cell invasion and metastasis (43).
In an NK-cell lymphoma cell line NK92, miRNA-494-3p was also found to down-regulate PTEN, which activated AKT in accordance with previous reports (39). Moreover, miRNA-494-3p worked in coordination with the EBV-encoded miRNA-BART20-5p, which inhibited the T-bet-PTEN pathway, with subsequent upregulation of AKT and suppression of TP53 (39). Antagomir to miRNA-494-3p may serve as a potential target of therapy of NKTCL (39).

MiRNA-150
MiRNA-150 as a key regulator of the differentiation and activation (44) of immune cells, such as B-, T-, and NKlymphocytes (45), abnormally expressed in solid (46) and hematological malignancies (44). MiRNA-150 was found to be apparently lower in lymphoma cell lines and primary lymphoma specimens compared with normal NK cells, while no significant difference was found between resting and activated NK cells (24). Furthermore, miRNA-150 down regulated PIK3AP1 and AKT2, which were part of the PI3K-AKT pathway and upregulated Bim and p53. MiRNA-150 led to cancer cell anti-apoptosis and immortality, as pAKT ser473/4 acted on telomerase via phosphorylation of hTERT (24). In addition, miRNA-150 down regulated DKC1, which functioned in regulating pseudouridine in RNA and the telomerase RNA subunit hTR in NKTCL cells (24,47). MiRNA-150 provides novel strategy upstream of AKT in the treatment of NKTCL (24).

MiRNA-223
MiRNA-223 is strongly expressed in the bone marrow and bone marrow cells but is absent in B-and T-lymphocytes (48). In resting NK cells, miRNA-223 downregulated in the case of cytokine activation and controls GzmB translation in resting NK cells (49). Overexpression of miRNA-223 can decrease cancer cell proliferation (50,51). For instance, miRNA-223 expression was reported to be lower in CD19 + lymphocytes in patients with mantle cell lymphoma compared with healthy donors (50). In NKTCL cells, overexpression of miRNA-223 is associated with cell differentiation (52). Positive regulatory domain containing I (PRDM1), a tumor suppressor gene in NK cell, was directly downregulated by miRNA-223 in NKTCL patient samples and NKTCL cell lines (53). All miRNA-223positive samples from patients with NKTCL showed EBV infection, which implied that EBV infection may be responsible for miRNA-223 overexpression (53).

MiRNA-16
Members of the miRNA-16 family function as tumor suppressors in a number of cancers via the regulation of the cell apoptosis pathway (54) and the cell cycle (55). In NK-cell lymphoma cell lines and primary tissue, miRNA-16 was found to be under expressed (56). MiRNA-16 and SAHA shared common

EBV-Encoded MiRNAs
Since NKTCL is an EBV associated lymphoma, researchers illustrated that EBV infection promoted the progression from a lesion into NKTCL (33,61,62) via the regulation of 44 microRNAs (59). The most common outcome of EBV infection was viral latency, including type I, II and III (63). Latency I was reported to only express characterized EBNA-1, while latency II expressed EBNA-1 as well as LMP-1 and 2. Moreover, latency III with B cell infection expressed all EBNAs and LMPs (64). Furthermore, latency patterns were distinct in different malignant subtypes due to the expression of subsets of the latent genes (65,66).
These studies have implications in the mechanisms of lymphomagenesis, and future experiments should be directed at the investigation of the role of EBV miRNAs and their regulation of cellular targets. The EBV-encoded miRNAs and their identified target genes are listed in Table 2 and associations are shown in Figure 2.
MiRNA-20, miRNA-26a, miRNA-92, miRNA-103, and miRNA-181 were shown to be overexpressed in patients of NKTCL (23). Moreover, miRNA-424 (38) and miRNA-16 (56) were shown to be under-expressed in NK-cell lymphoma cell lines and tumor tissue. The expression of miRNA-221 in the serum of NKTCL patients might be a prognostic factor since high expression leads to a poorer overall survival (OS) (78).
MiRNA-15a was reported to inhibit the cell cycle by blocking G1/S progression in NK-cell lymphoma cell lines (58). Specifically, miRNA-15a upregulated MYB and cyclin D1 which were essential for the proliferation of NK-cell lymphoma cells (58).
As a tumor suppressor, miRNA-34a (79) was found to be hypermethylated in both myeloma and lymphoma cell lines (80). Furthermore, in lymphoma primary patient samples,

MiRNA Targets Targets function References
Upregulation Downregulation
methylation of miRNA-34a was found to be more frequently in NKTCL than in B-or T-cell lymphoma (80). MiRNA-146a also exhibited hypermethylation in NKTCL, and down-regulated its target gene TRAF6 and NF-κB signaling pathway (60). In clinical study, low miRNA-146a expression was an independent poor prognostic factor.
In summary, we found that dysregulation of miRNAs might be a key feature of the pathogenesis of NKTCL. Aberrant expression of miRNAs might affect the AKT, NF-κB and cell cycle signaling pathways to regulate cell function. The signaling pathway model has been integrated in Figure 1. Hypermethylation is another way by which cell function is regulated. These findings provide new thought about the pathogenesis of NKTCL.

LncRNAs
LncRNAs are a group of RNAs >200 nucleotides in length that regulate gene expression by transcriptional and posttranscriptional destabilization (81,82). LncRNAs have great potential value in the pathogenesis, diagnosis, treatment and prognosis of malignant tumors (83,84). Baytak et al. (85) conducted whole transcriptome sequencing (WTS) analysis on NKTCL cases, normal NK-cells and NK-cell lymphoma cell lines. They revealed 166 lncRNAs with more than 1.5-fold overexpression, such as RAB30-AS1, ARAP-AS1 and PRMT5-AS1 which may have biological function on cell growth.
LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched transcript 2 (NEAT2), is discovered as a predictive marker for metastasis and survival in early-stage, non-small cell lung cancer (94). The high expression of MALAT1 have been found in various cancer types (95,96) and predicted metastasis or poor prognosis (97,98). LncRNA MALAT1 was highly expressed in NKTCL, but in the absence of expression, inferior OS is observed. MALAT1 was important in sustaining PRC2-induced H3K27me3, which led to the subsequent activation of BMI1 which predicted the clinically aggressive behaviors in NKTCL (99,100).
The functions and regulations of lncRNAs were not isolated. Positive regulatory domain containing I (PRDM1), which acted as a tumor suppressor gene, was silenced in NKTCL as previously mentioned (7). PRDM1α regulated 212 lncRNAs (169 upregulation and 103 downregulation). Among them, MIRNA-155HG and TERC may be an indirect target of PRDM1 in NK-cell lymphoma cells. However, biological functions of most lncRNAs regulated by PRDM1 in NKTCL remained unknown, and further studies should involve functional characterization (85).

CONCLUSION
With the development of next-generation sequencing and bioinformatics, non-coding RNAs have shown promising value in tumor research in recent years. However, the focus on B-cell and T-cell lymphoma encompasses much more than NKTCL. This article summarizes the function of significantly differentially expressed hotspot non-coding RNAs that contribute to the pathogenesis, diagnoses, treatment and prognosis of NKTCL. The detailed mechanisms and the function of the noncoding RNAs mentioned above still remain to be clarified. Further connections among these non-coding RNAs can be supplemented. Moreover, research on novel non-coding RNAs such as piwi RNAs, circular RNAs and tiRNAs that are associated with NKTCL is needed. Clinical research of thoroughly studied non-coding RNAs could be performed and lead to the early diagnoses of NKTCL and could be useful in drug resistance or targeted therapy.

AUTHOR CONTRIBUTIONS
MZ and MM designed the study. MM collected data and wrote the manuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS
I would like to show gratitude to all members in oncology department of the First Affiliated Hospital of Zhengzhou University.